Method for protecting chickens and hogs from pasteurella

ABSTRACT

IN WHICH R is a member of the group consisting of hydrogen and a tetrahydrofurylacetylamino radical, TO THE FOWL OR HOGS TO BE PROTECTED.   A method of protecting fowl and hogs from Pasteurella which comprises administering an effective dose of a compound of the formula

United States Patent Ueno et al.

[ Sept. 18, 1973 METHOD FOR PROTECTING CHICKENS AND HOGS FROMPASTEURELLA Inventors: Ryuzo Ueno, Nishinomiya; Shota Watanabe,Takarazuka; Sotoyuki Ota, Nishinomiya, all of Japan Kabushiki KaishaUeno Seiyaku Oyo Kenkyujo, Osaka, Japan Filed: Jan. 5,1971

Appl. No.: 104,156

Assignee:

U.S. Cl. 424/272 Int. Cl A6lk 27/00 Field of Search 424/272; 260/240 A,

References Cited UNITED STATES PATENTS 4/l97l Hirao et al 424/272Primary ExaminerSam Rosen Att0rneySherman & Shalloway [57] ABSTRACT Amethod of protecting fowl and hogs from Pasteurella which comprisesadministering an effective dose of a m es in which R is a member of thegroup consisting of hydrogen and a tetrahydrofurylacetylamino radical,to the fowl or hogs to be protected.

4 Claims, No Drawings METHOD FOR PROTECTING CHICKENS AND HOGSFROMPASTEURELLA ill.

in which R is a member of the group consisting of hydrogen and atetrahydrofurylacetylamino radical.

Domestic fowl and animals are susceptible to infections with variousgerms during their growth, and are suceptible to being taken ill. Whenfowl or chickens are infected with Pasteurella multocida there is a highmortality rate. Also hogs are apt to beinfected with microorganisms ofthe Pasteurella species, for example, Pasteurella multocida, and fallinto subacute or chronic disease. It is now found that theabove-specified compounds exhibit very efi'ective preventive andtherapeutic action to the fowl and hogs which are infected withmicroorganisms of the Pasteurella species.

The specified compounds exhibit remarkably effective antibacterialaction against microorganisms of the Pasteurella species, e.g., Past.hemolytica, Past. multocida, Past. pestis and Past. tularensis, interalia, Past. mulrocida.

The effective doses of the compounds can be used in combination withvarious known carriers and/or adjuvants in accordance with the acceptedpractice. In a particularly preferred embodiment, a powder medicinecontaining 2 to 10 percent by weight of either of the specifiedcompounds as the effective ingredient, is mixed with a filling agentsuch as corn meal, lactose, starch, talc, etc., and is added to the feedof chickens and hogs. It may also be conveniently administered in theform of liquid medicine, together with a wetting agent, solubilizingagent, surface active agent, e.g., polyoxyethylene glycol, CMC,glycerine, etc., or suspension such as acacia.

The effective dose of the compound ranges normally from 10 to 200 mg,preferably 50 180 mg. When it is added to chicken or hog feed,respectively 0.005 0.01 wt. percent, and 0.01 0.02 wt. percent, based onthe weight of the feed, is conveniently used.

The period of administration of the compound varies, depending on theseriousness of symptoms, while normally continuous administration ofapproximately 7 to 30 days is sufficient. The effect of the compoundbecomes appreciable from the 4th to 5th day after the firstadministration. Thus the compounds very effectively protect chickens andhogs from diseases caused by microorganisms of the Pasteurella species.

The melting points and the minimum inhibiting concentrations (lMC) invitro against Pasteurella multocida of the subject compounds are shownin Table 1 below. From the data it should be understood that thecompounds possess very strong antibacterial action.

TABLE L-THE MINIMUM INHIBITING CONCENTRATION (IN VI'IRO) i i R: 00011\0/ Appearance: yellow, needle-like crystal.

Appearance: yellow,

columnar crystal.

Pasleurella multocida.. 4.0

The minimum inhibiting concentration was determined in the followingmanner. To tryptosoybouillon (the tryptosoybouillon being obtained bydissolving 17 g of tripton, 3 g of soypeptone, 2.5 g of glucose, 2.5g ofpotassium hydrogenphosphate, and 5 g of sodium chloride in 1 liter ofwater, which had a pH of 7.3), cows serum was added in an amount of 10percent by volume based on the volume of the tryptosoybouillon.Separately, the compound of the invention was dissolved indimethylsulfoxide, and the solution was added to the above medium at aratio of 2 volume percent based on the latter, i.e., 25 'y/ml. of themedium. The medium was diluted to various concentrations in conventional manner. To 2 ml of media containing various concentrations ofactive ingredient a drop of the germ liquid (cultured for 20 hours in aninitially prepared medium free of the active compound of the invention)were inoculated with a syringe and incubated at 37C for 48 hours.Thereafter the turbidity of each medium was checked to judge if thegerms propagated.

It has been proven through the experiments with mice that the subjectcompounds are extremely stable and show substantial activity when theyare administered to animals. When the compounds are orally administeredto C3l-l type mice of approximately 18 grams each in body weight, whichwere artificially infected with Pasteurella multocida inoculated intotheir abdominal cavities, the median effective doses (EDSO) were asgiven in Table 2 below. From the table, it can be clearly understoodthat the effect of the compounds is notably better than that ofanalogous compounds having NH or -NHCOCH at the second position of theoxadiazole ring.

Administered dose (m g./kg.)

Compound 800 400 200 100 50 Subject compound Survived mice/tested micc3/5 1/5 /5 E1); (mg/kg.) 168 l v v O k-I\ l H H I! a, O 0 CH C O Subjectcompound Survived m icc/tcstcd mice t. 5/5 5/5 1/5 Elm (mg/kg.) 68 o N NII It =1 l u 4 I OQN o CH=(,-- O -i\HCOCHz- I6I Control compoundSurvivcd micc/tcstcd mic0 l l 5/5 2/5 0/5 EDso (mg/kg.) 466 O NN l H ILI t I y. l w t O CH O \H.

Control compound Survived micc/testcd micc 0/5 O/5 EDsu (mg/kg.) 800 O\l\ l H J'NIICOCII3 The manner of measurement and determination of EDSOwas as follows: Pasteurella multocida p-l059 was cultured intryptosoybouillon containing 5 percent of rabbits blood for 20 hours at37C., and 0.2 ml each of 10" dilution was inoculated into the abdominalcavity of a mouse (4 weeks old, weighing 18 g).

Immediately after the inoculation, the dosage of each compound to beadministered was ground in an agate mortar, suspended with acacia, and0.4 ml thereof per mouse was orally administered. One test groupconsisted of five mice.

From the number of mice surviving after 5 days from an inoculation, EDSOwas calculated according to the Behrens-Kaerber method.

The compounds of the subject invention are further found to possessexcellent properties as follows:

Generally nitrofuran derivatives are sparingly soluble in water, andwhen orally administered to animals, their absorption through theintestines is low. Consequently, only a minor percentage of theadministered compound migrates into the blood of the animals. On thecontrary, the compounds of the invention have higher solubilities inwater than known nitrofuran derivatives, and can migrate into blood ofthe administered animals at higher concentrations. In Table 3 below, thesolubilities of subject compounds, and their concentrations in blood ofrats which had been orally administered with the compounds are comparedwith the similar data of analogous compounds.

TABLE 3.CONCENTRATION IN RATS BLOOD (y/ml.)

Hours after the compound's udministration Solubil- Compound ity 0 2 3 824 48 Subject compound 28. 2 0 0.15 0. l2 0. 08 0. 02 0. ()16 Subjectcompound 11.0 0 6. 3 5. 6 2.1 0. 06 0. 06

0N -cH=o- \HCOCH H \O/ \O/ \O/ Control compound 3. 2 0 0.03 0. 027 0. 040. 024 0. 016

N--N 'l i 11/ H H v O I\ O CH -C NH Control compound 7.5 0 0.025 0.020.016 0.016 0.0l6

O NN The concentration in blood was measured as follows:

Each of the compounds wassuspended in 5 percent acacia at a ratio ofmg/ml, and orally administered to male rats weighing approximately 150 geach, at a dose of 100 mg/kg. At 2, 3, 8, 24, and 48 hours after theadministration, the rats were anesthetized with ether, and their bloodwas extracted from the heart with a syringe. One test group consisted ofthree rats. The blood was centrifuged after coagulation, and the serumas the supernatant was separated and stored at C.

The concentration of each compound in the serum was determined by meansof bioassay. The bioassay medium (which was prepared by dissolving 0.6percent of peptone, 0.3 percent of yeast extract, 0.15 percent of meatextract, and 1 percent of agar in distilled water, and adjusting the pHof the solution to 7.0) was sterilized and maintained at 50C. To suchmedium, 2ml of 1 percent NaNO 3.8 ml of 0.1 percent Methylene Blue, and0.2 ml of test germ liquid (which was prepared by culturing Escherichiacoli 8057 in normal bouillon for 20 hours at 37C.), per 100 m] of themedium, were added and mixed, and 2 ml samples of the mixture werepoured into test tubes of 7 mm in inner diameter. The culture medium wasallowed to coagulate by standing at room temperature for approximately30 minutes. On each medium in the test tubes, 0.2 ml each of standarddilution of the test compound at various concentrations (16, 8, 4, 2, 1,0.5, 0.25, 0.125, 0.0062, 0.031, 0.016, and 0.008 'y/ml was superposed,(one sample was superposed in three test tubes.) Thereafter the compoundwas allowed to penetrate into the medium while being kept in arefrigerator for 2 hours, followed by an overnight incubating at 37C.The length of the inhibition zone, in which the Methylene Blue was notreduced, in all the test tubes was measured to draw a standard curve,and from the standard curve the concentration of the compound in eachserum was calculated.

Furthermore, the median lethal doses (LDSO) of the subject compounds areextremely high thereby persuasively demonstrating the very high safetyof the compounds.

The median lethal doses of the subject compounds suspended in 5 percentaqueous solution of acacia, when orally administered to C3H type mice ofeach approximately 18 g in body weight, were as given in Table 4 below.The data prove the very low toxicity of the compounds studied.

As described above, the subject compounds not only exhibit substantialantibacterial activity in vitro, but also prevent Pasteurella multocidainfection when administered to chickens and hogs, much more effectivelythan the analogous nitrofuran derivatives. Furthermore, the compoundsproduce immediate effect, have very low toxicity, and therefore havebroad safety margins. Thus the compounds can very effectively protectchickens and hogs from the infection with Pasteurella. I

EXAMPLE 1 The subject compounds were added to the feed of 14 day-oldchickens. Each test group consisted of 10 chickens On the second day ofthe administration, 0.2 ml of 10 dilute inoculum of Pasteurellamultocida cultured in tryptosoy broth containing 10 percent of rabbitsblood for 24 hours was inoculated into each chicken by means ofintramuscular injection in the left leg. The administration of subjectcompound was continued for 5 days following the inoculation, and thecompound's effect was evaluated by the ratio of chickens that survived.The results were as shown in Table 5 below. It is seen that the chickensfed with the feed containing 0.0l percent of the subject compounds wereprotected from infection and survived by 100 percent and percent, whilethose fed with the feed containing 0.005 percent of either of thecompounds, by 60 percent. In clear contrast, the chickens administeredwith known control compounds were all infected and died.

EXAMPLE 2 Young mongrel pigs of Berkshire origin which were to be soldas soon as their body weights reached 90 Kg each were continuouslyadministered with the subject compound contained in their feed in anamount of 0.015 percent.

Thirty pigs were selected from those borne on the same day from foursows and divided into three test groups, each group consisting of 10pigs.

The test started on the 73rd day from their birth, and the number ofdays required for them to reach 90 Kg was studied as was the feedconversion and lung lesion, of the pigs. It was confirmed as the resultof the studies that the pigs which were administered with the subjectcompounds showed excellent achievement over the control groups fed withcontrol compounds.

It was confirmed that the number of days required for the pigs to weigh90 Kg was less with the test groups fed with the subject compounds. Themacro examination of the lugs of the pigs upon pathological anatomy ofthe pigs after slaughtering confirmed the substanial effect of thesubject compounds, since the lesions in the lungs were less with thetreated groups than with the control groups.

Furthermore, upon bacteriological study of the pigs,

microorganisms of the Pasreurella species were de-' tected from the pigsof the control groups, but none was detected from the lungs of the testgroups administered with the subject compounds.

TABLE 6 Number of Addition Number survived to feed, of tested chickens}Compound percent chickens percent Subject compound 0. 01 10 100 0. 00510 60 U I O NN OzN-L J-CH=C m J Subject compound 0. 01 10 90 0. 005 1o60 i 0 NN l l IL L I O:N CH=C NHCOCH2 Control compound 0. 01 10 O 0.00510 0 a W W O:N'\ )CH=C i /NH2 0 0 Control compound 0. 1 10 0 0.005 10 0W Oz-N LCII=C I LNHCOCH:

1 After 5 days of administration.

TABLE 6 Subject compound Control compound Basic iced b NN b i it u l l lO:N CH:C R 02N' -CH=C NH2 R211 H I RINHCOCIL 0 Quantity added to thefeed. percent 0.015 0.015 0.015 0 Number of tested pigs 10(96, 6'4)10(96, 6'4) 10(96, 6'1) (96, 6' Average number of days from birth:

Beginning 73 73 End 168;];6. 2 1695. 0 172;|;7. 5 183=|=7. 4 Number ofdays required [or increasing pi weight to 90 kg. each 955:6. 3 065:6. 8995:7. 5 110:1:1 Ai'erage body weight (kg):

Beginning. 20. 63:3. 5 20. :3. 8 20. 513. 6 20. 51:4. 2 Ending do 90.290.2 90.1 90- Average body weight increase per day 4.) 732. G3 726. 04703.03 634. 54 Average feed consumption per pig (kg) 211. 68 218.60219.94 242. 90 Feed conversion 3. 04 3. l4 3. 16 3. 4 Lesion in lung:

Serious 1 2 4 6 Medium 1 1 2 1 Slight... 3 1 1 0 Normal 5 6 2 3 Weclaim: 55 wherein R is a member of the group consisting of hy- 1. Amethod of protecting chickens and hogs from drogen and atetrahydro-furylacetylamino radical. microor anisms of the Pasteurella secies which comg 2. The method of claim 1, wherein the effective doseprises orally administering to said chickens and hogs an ranges from 10to ZOO-mg. effective dose of a compound of the formula,

3. The method of claim 1, wherein the compound is administered in theanimal feed. I 4. The method of claim 1, wherein the microorgan- 0,N011:0 isms of the Pasteurella species are Pasteurella mul- 0 tocida.

2. The method of claim 1, wherein the effective dose ranges from 10 to200-mg.
 3. The method of claim 1, wherein the compound is administeredin the animal feed.
 4. The method of claim 1, wherein the microorganismsof the Pasteurella species are Pasteurella multocida.